Dental biofilms are complex communities composed largely of harmless bacteria. Certain pathogenic species including Streptococcus mutans (S. mutans) can become predominant when host factors such as dietary sucrose intake imbalance the biofilm ecology. Current approaches to control S. mutans infection are not pathogen-specific and eliminate the entire oral community along with any protective benefits provided. Here, we tested the hypothesis that removal of S. mutans from the oral community through targeted antimicrobial therapy achieves protection against subsequent S. mutans colonization.
Controlled amounts of S. mutans were mixed with S. mutans-free saliva, grown into biofilms and visualized by antibody staining and cfu quantization. Two specifically-targeted antimicrobial peptides (STAMPs) against S. mutans were tested for their ability to reduce S. mutans biofilm incorporation upon treatment of the inocula. The resulting biofilms were also evaluated for their ability to resist subsequent exogenous S. mutans colonization.
S. mutans colonization was considerably reduced (9 ± 0.4 fold reduction, P=0.01) when the surface was preoccupied with saliva-derived biofilms. Furthermore, treatment with S. mutans-specific STAMPs yielded S. mutans-deficient biofilms with significant protection against further S. mutans colonization (5 minutes treatment: 38 ± 13 fold reduction P=0.01; 16 hours treatment: 96 ± 28 fold reduction P=0.07).
S. mutans infection is reduced by the presence of existing biofilms. Thus maintaining a healthy or “normal” biofilm through targeted antimicrobial therapy (such as the STAMPs) could represent an effective strategy for the treatment and prevention of S. mutans colonization in the oral cavity and caries progression.
targeted antimicrobial therapy; antimicrobial peptide; biofilm; Streptococcus mutans; protective colonization; caries
Streptococcus mutans has been implicated as the major acid-producing (cariogenic) bacterium. Dietary sugars and other factors may cause an imbalance of oral microflora that enables S. mutans to become dominant in the multi-species biofilms on the tooth surface, which could lead to dental caries. The application of broad-spectrum antimicrobials often results in re-colonization and re-dominance of S. mutans within oral flora, while in contrast, therapies capable of selective elimination of S. mutans from oral microbial communities may help to re-establish the normal flora and provide long-term protection. C16G2, a novel synthetic antimicrobial peptide with specificity for S. mutans, was found to have robust killing efficacy and selectivity for S. mutans in vitro. A subsequent pilot human study found that a single application of C16G2 in the oral cavity (formulated in a mouthrinse vehicle) was associated with a reduction in plaque and salivary S. mutans, lactic acid production, and enamel demineralization during the entire 4-day testing period. C16G2 is now being developed as a new anticaries drug.
microbial ecology; microbiology; microbial genetics; caries; dental biofilm; microbiota
Streptococcus mutans, the major etiological agent of dental caries, has a measurable impact on domestic and global health care costs. Though persistent in the oral cavity despite conventional oral hygiene, S. mutans can be excluded from intact oral biofilms through competitive exclusion by other microorganisms. This suggests that therapies capable of selectively eliminating S. mutans while limiting the damage to the normal oral flora might be effective long-term interventions to fight cariogenesis. To meet this challenge, we designed C16G2, a novel synthetic specifically targeted antimicrobial peptide with specificity for S. mutans. C16G2 consists of a S. mutans-selective ‘targeting region’ comprised of a fragment from S. mutans competence stimulation peptide (CSP) conjoined to a ‘killing region’ consisting of a broad-spectrum antimicrobial peptide (G2). In vitro studies have indicated that C16G2 has robust efficacy and selectivity for S. mutans, and not other oral bacteria, and affects targeted bacteria within seconds of contact.
In the present study, we evaluated C16G2 for clinical utility in vitro, followed by a pilot efficacy study to examine the impact of a 0.04% (w/v) C16G2 rinse in an intra-oral remineralization/demineralization model.
Results and Conclusions
C16G2 rinse usage was associated with reductions in plaque and salivary S. mutans, lactic acid production, and enamel demineralization. The impact on total plaque bacteria was minimal. These results suggest that C16G2 is effective against S. mutans in vivo and should be evaluated further in the clinic.
Antimicrobial; Antimicrobial peptide; Caries; Demineralization; Dental plaque; Lactic acid; Mouth rinse; Oral therapeutic; Selective antibiotic; Selective therapeutic; Specifically targeted antimicrobial peptide; Streptococcus mutans; Targeted antimicrobial
Streptococcus mutans is the primary cariogen that produces several virulence factors that are modulated by a competence-stimulating peptide (CSP) signaling system. In this study, we sought to determine if proteases produced by early dental plaque colonizers such as Streptococcus gordonii interfere with the subsequent colonization of S. mutans BM71 on the existing streptococcal biofilms. We demonstrated that S. mutans BM71 colonized much less efficiently in vitro on streptococcal biofilms than on Actinomyces naeslundii biofilms. Several oral streptococci, relative to A. naeslundii, produced proteases that inactivated the S. mutans CSP. We further demonstrated that cell protein extracts from S. gordonii, but not from A. naeslundii, interfered with S. mutans BM71 colonization. In addition, S. mutans BM71 colonized more efficiently on the sgc protease knockout mutant of S. gordonii than on the parent biofilms. In conclusion, proteases of early colonizers can interfere with subsequent colonization by S. mutans in vitro.
Streptococcus mutans; Streptococcus gordonii; Actinomyces naeslundii; biofilm; protease; quorum sensing
Active immunization with Streptococcus mutans glucan binding protein B (GBP-B) has been shown to induce protection against experimental dental caries. This protection presumably results from continuous secretion of salivary antibody to GBP-B, which inhibits accumulation of S. mutans within the oral biofilm. The purpose of this study was to explore the influence of short-term (9- or 24-day) passive oral administration of antibody to S. mutans GBP-B on the longer-term accumulation and cariogenicity of S. mutans in a rat model of dental caries. Preimmune chicken egg yolk immunoglobulin Y (IgY) or IgY antibody to S. mutans GBP-B was supplied in lower (experiment 1) and higher (experiment 2) concentrations in the diet and drinking water of rats for 9 (experiment 1) or 24 (experiment 2) days. During the first 3 days of IgY feeding, all animals were challenged with 5 × 106 streptomycin-resistant S. mutans strain SJ-r organisms. Rats remained infected with S. mutans for 78 days, during which rat molars were sampled for the accumulation of S. mutans SJ-r bacteria and total streptococci. Geometric mean levels of S. mutans SJ-r accumulation on molar surfaces were significantly lower in antibody-treated rats on days 16 and 78 of experiment 2 and were lower on all but the initial (day 5) swabbing occasions in both experiments. Relative to controls, the extent of molar dental caries measured on day 78 was also significantly decreased. The decrease in molar caries correlated with the amount and duration of antibody administration. This is the first demonstration that passive antibody to S. mutans GBP-B can have a protective effect against cariogenic S. mutans infection and disease. Furthermore, this decrease in infection and disease did not require continuous antibody administration for the duration of the infection period. This study also indicates that antibody to components putatively involved only in cellular aggregation can have a significant effect on the incorporation of mutans streptococci in dental biofilm.
The caries incidence at various levels of Streptococcus mutans infection was analyzed in a control group and a test group. In the control group, the incidence of caries and the duration of S. mutans infection were significantly correlated. In the test group, the S. mutans infection was suppressed by antimicrobial measures when the number of S. mutans exceeded 250 X 10(3) CFU per ml of saliva. The results illustrate that the level and duration of the S. mutans infection are strongly correlated to the incidence of caries. The findings support the concept of S. mutans as a key cariogenic microorganism and illustrate the value of antimicrobial treatment in the prevention of caries.
Streptococcus mutans, the primary etiologic agent of dental caries, possesses a series of virulence factors associated with its cariogenicity. Alternatives to traditional antimicrobial treatment, agents selectively inhibiting the virulence factors without necessarily suppressing the resident oral species, are promising. The anticariogenic properties of tea have been suggested in experimental animals and humans. Tea polyphenols, especially epigallocatechin gallate (EGCg), have been shown to inhibit the growth and glucosyltransferases activity of S. mutans. However, their effects on biofilm and cariogenic virulence factors of oral streptococci other than glucosyltransferases have not been well documented. In this study, we investigated the biological effect of EGCg on the virulence factors of S. mutans associated with its acidogenicity and acidurity. The antimicrobial effects of EGCg on S. mutans biofilm grown in chemically defined medium were also examined. EGCg inhibited growth of S. mutans planktonic cells at an MIC of 31.25 μg/ml and a minimal bactericidal concentration (MBC) of 62.5 μg/ml. EGCg also inhibited S. mutans biofilm formation at 15.6 μg/ml (minimum concentration that showed at least 90% inhibition of biofilm formation) and reduced viability of the preformed biofilm at 625 μg/ml (sessile MIC80). EGCg at sub-MIC levels inhibited acidogenicity and acidurity of S. mutans cells. Analysis of the data obtained from real-time PCR showed that EGCg significantly suppressed the ldh, eno, atpD, and aguD genes of S. mutans UA159. Inhibition of the enzymatic activity of F1Fo-ATPase and lactate dehydrogenase was also noted (50% inhibitory concentration between 15.6 and 31.25 μg/ml). These findings suggest that EGCg is a natural anticariogenic agent in that it exhibits antimicrobial activity against S. mutans and suppresses the specific virulence factors associated with its cariogenicity.
Unlike many pathogens are foreign invaders, oral “pathogens” such as Streptococcus mutans are part of the “normal” oral microbial flora. While they express certain pathogenic properties, the balance of synergistic and antagonistic interactions determines whether these çommensal pathogens cause damage or not. Recognition of these microbial ecology based pathogeneses argues for new strategies for disease treatment and prevention.
Probiotics, potentially beneficial live bacteria or yeasts, have been used to combat dental caries. This includes the application of S. mutans types that cannot produce acids or other bacteria that interfere with the pathogenic effects of S. mutans. While these approaches show therapeutic effects against S. mutans experimentally, the conversion into commercial products remains a challenge, due to safety and shelf life issues. New high-tech approaches, such as quorum sensing interference of pathogenic bacteria or targeted antimicrobial therapies, offer novel ways to achieve probiotic effects against dental caries.
The specifically targeted antimicrobial peptide (STAMP) C16G2 was developed to target the cariogenic oral pathogen Streptococcus mutans. Because the design of this peptide was novel, we sought to better understand the mechanism through which it functioned. Compared to antimicrobial peptides (AMPs) with wide spectra of activity, the STAMP C16G2 has demonstrated specificity for S. mutans in a mixed-culture environment, resulting in the complete killing of S. mutans while having minimal effect on the other streptococci. In the current study, we sought to further confirm the selectivity of C16G2 and also compare its membrane activity to that of melittin B, a classical toxic AMP, in order to determine the STAMP's mechanism of cell killing. Disruption of S. mutans cell membranes by C16G2 was demonstrated by increased SYTOX green uptake and ATP efflux from the cells similar to those of melittin B. Treatment with C16G2 also resulted in a loss of membrane potential as measured by DiSC(3)5 fluorescence. In comparison, the individual moieties of C16G2 demonstrated no specificity and limited antimicrobial activity compared to those of the STAMP C16G2. The data suggest that C16G2 has a mechanism of action similar to that of traditional AMPs and kills S. mutans through disruption of the cell membrane, allowing small molecules to leak out of the cell, which is followed by a loss of membrane potential and cell death. Interestingly, this membrane activity is rapid and potent against S. mutans, but not other noncariogenic oral streptococci.
Hydroxychavicol isolated from the chloroform extraction of aqueous extract of Piper betle leaves showed inhibitory activity against oral cavity pathogens. It exhibited an inhibitory effect on all of the oral cavity pathogens tested (MICs of 62.5 to 500 μg/ml) with a minimal bactericidal concentration that was twofold greater than the inhibitory concentration. Hydroxychavicol exhibited concentration-dependent killing of Streptococcus mutans ATCC 25175 up to 4× MIC and also prevented the formation of water-insoluble glucan. Interestingly, hydroxychavicol exhibited an extended postantibiotic effect of 6 to 7 h and prevented the emergence of mutants of S. mutans ATCC 25175 and Actinomyces viscosus ATCC 15987 at 2× MIC. Furthermore, it also inhibited the growth of biofilms generated by S. mutans and A. viscosus and reduced the preformed biofilms by these bacteria. Increased uptake of propidium iodide by hydroxychavicol-treated cells of S. mutans and A. viscosus indicated that hydroxychavicol probably works through the disruption of the permeability barrier of microbial membrane structures. Hydroxychavicol also exhibited potent antioxidant and anti-inflammatory activities. This was evident from its concentration-dependent inhibition of lipid peroxidation and significant suppression of tumor necrosis factor alpha expression in human neutrophils. Its efficacy against adherent cells of S. mutans in water-insoluble glucan in the presence of sucrose suggests that hydroxychavicol would be a useful compound for the development of antibacterial agents against oral pathogens and that it has great potential for use in mouthwash for preventing and treating oral infections.
Within the repertoire of antibiotics available to a prescribing clinician, the majority affect a broad range of microorganisms, including the normal flora. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. To address this problem, our laboratory has recently developed a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs), based on the fusion of a species-specific targeting peptide domain with a wide-spectrum antimicrobial peptide domain. In the current study, we focused on achieving targeted killing of Streptococcus mutans, a cavity-causing bacterium that resides in a multispecies microbial community (dental plaque). In particular, we explored the possibility of utilizing a pheromone produced by S. mutans, namely, the competence stimulating peptide (CSP), as a STAMP targeting domain to mediate S. mutans-specific delivery of an antimicrobial peptide domain. We discovered that STAMPs constructed with peptides derived from CSP were potent against S. mutans grown in liquid or biofilm states but did not affect other oral streptococci tested. Further studies showed that an 8-amino-acid region within the CSP sequence is sufficient for targeted delivery of the antimicrobial peptide domain to S. mutans. The STAMPs presented here are capable of eliminating S. mutans from multispecies biofilms without affecting closely related noncariogenic oral streptococci, indicating the potential of these molecules to be developed into “probiotic” antibiotics which could selectively eliminate pathogens while preserving the protective benefits of a healthy normal flora.
A biofilm is a complex community of microorganisms that develop on surfaces in diverse environments. The thickness of the biofilm plays a crucial role in the physiology of the immobilized bacteria. The most cariogenic bacteria, mutans streptococci, are common inhabitants of a dental biofilm community. In this study, DNA-microarray analysis was used to identify differentially expressed genes associated with the thickness of S. mutans biofilms.
Comparative transcriptome analyses indicated that expression of 29 genes was differentially altered in 400- vs. 100-microns depth and 39 genes in 200- vs. 100-microns biofilms. Only 10 S. mutans genes showed differential expression in both 400- vs. 100-microns and 200- vs. 100-microns biofilms. All of these genes were upregulated.
As sucrose is a predominant factor in oral biofilm development, its influence was evaluated on selected genes expression in the various depths of biofilms. The presence of sucrose did not noticeably change the regulation of these genes in 400- vs. 100-microns and/or 200- vs. 100-microns biofilms tested by real-time RT-PCR.
Furthermore, we analyzed the expression profile of selected biofilm thickness associated genes in the luxS- mutant strain. The expression of those genes was not radically changed in the mutant strain compared to wild-type bacteria in planktonic condition. Only slight downregulation was recorded in SMU.2146c, SMU.574, SMU.609, and SMU.987 genes expression in luxS- bacteria in biofilm vs. planktonic environments.
These findings reveal genes associated with the thickness of biofilms of S. mutans. Expression of these genes is apparently not regulated directly by luxS and is not necessarily influenced by the presence of sucrose in the growth media.
Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (P<0.05). Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other organisms. Our data provide insights about how S. mutans optimizes its metabolism and adapts/survives within the mixed-species community in response to a dynamically changing environment. This reflects the intricate physiological processes linked to expression of virulence by this bacterium within complex biofilms.
Streptococcus mutans, the predominant bacterial species associated with dental caries, can enter the bloodstream and cause infective endocarditis. The aim of this study was to investigate S. mutans biofilm formation and adherence to endothelial cells induced by human fibrinogen. The putative mechanism by which biofilm formation is induced as well as the impact of fibrinogen on S. mutans resistance to penicillin was also evaluated. Bovine plasma dose dependently induced biofilm formation by S. mutans. Of the various plasma proteins tested, only fibrinogen promoted the formation of biofilm in a dose-dependent manner. Scanning electron microscopy observations revealed the presence of complex aggregates of bacterial cells firmly attached to the polystyrene support. S. mutans in biofilms induced by the presence of fibrinogen was markedly resistant to the bactericidal effect of penicillin. Fibrinogen also significantly increased the adherence of S. mutans to endothelial cells. Neither S. mutans cells nor culture supernatants converted fibrinogen into fibrin. However, fibrinogen is specifically bound to the cell surface of S. mutans and may act as a bridging molecule to mediate biofilm formation. In conclusion, our study identified a new mechanism promoting S. mutans biofilm formation and adherence to endothelial cells which may contribute to infective endocarditis.
The combination of sucrose and starch in the presence of surface-adsorbed salivary α-amylase and bacterial glucosyltransferases increase the formation of a structurally and metabolically distinctive biofilm by Streptococcus mutans. This host-pathogen-diet interaction may modulate the formation of pathogenic biofilms related to dental caries disease. We conducted a comprehensive study to further investigate the influence of the dietary carbohydrates on S. mutans-transcriptome at distinct stages of biofilm development using whole genomic profiling with a new computational tool (MDV) for data mining. S. mutans UA159 biofilms were formed on amylase-active saliva coated hydroxyapatite discs in the presence of various concentrations of sucrose alone (ranging from 0.25 to 5% w/v) or in combination with starch (0.5 to 1% w/v). Overall, the presence of sucrose and starch (suc+st) influenced the dynamics of S. mutans transcriptome (vs. sucrose alone), which may be associated with gradual digestion of starch by surface-adsorbed amylase. At 21 h of biofilm formation, most of the differentially expressed genes were related to sugar metabolism, such as upregulation of genes involved in maltose/maltotriose uptake and glycogen synthesis. In addition, the groEL/groES chaperones were induced in the suc+st-biofilm, indicating that presence of starch hydrolysates may cause environmental stress. In contrast, at 30 h of biofilm development, multiple genes associated with sugar uptake/transport (e.g. maltose), two-component systems, fermentation/glycolysis and iron transport were differentially expressed in suc+st-biofilms (vs. sucrose-biofilms). Interestingly, lytT (bacteria autolysis) was upregulated, which was correlated with presence of extracellular DNA in the matrix of suc+st-biofilms. Specific genes related to carbohydrate uptake and glycogen metabolism were detected in suc+st-biofilms in more than one time point, indicating an association between presence of starch hydrolysates and intracellular polysaccharide storage. Our data show complex remodeling of S. mutans-transcriptome in response to changing environmental conditions in situ, which could modulate the dynamics of biofilm development and pathogenicity.
Dental caries occurrence is caused by the colonization of oral microorganisms and accumulation of extracellular polysaccharides synthesized by Streptococcus mutans with the synergistic influence of Lactobacillus spp. bacteria. The aim of this study was to determine ex vivo the antibacterial properties of ethanol extract of propolis (EEP), collected in Poland, against the main cariogenic bacteria: salivary mutans streptococci and lactobacilli. The isolation of mutans streptococci group bacteria (MS) and Lactobacillus spp. (LB) from stimulated saliva was performed by in-office CRT bacteria dip slide test. The broth diffusion method and AlamarBlue assay were used to evaluate the antimicrobial activity of EEP, with the estimation of its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The biochemical composition of propolis components was assessed. The mean MIC and MBC values of EEP, in concentrations ranging from 25 mg/mL to 0.025 mg/mL, for the MS and LB were found to be 1.10 mg/mL versus 0.7 mg/mL and 9.01 mg/mL versus 5.91 mg/mL, respectively. The exposure to an extract of Polish propolis affected mutans streptococci and Lactobacillus spp. viability, exhibiting an antibacterial efficacy on mutans streptococci group bacteria and lactobacilli saliva residents, while lactobacilli were more susceptible to EEP. Antibacterial measures containing propolis could be the local agents acting against cariogenic bacteria.
Initial attachment of the cariogenic Streptococcus mutans onto dental enamel is largely promoted by the adsorption of specific salivary proteins on enamel surface. Some phosphorylated salivary proteins were found to reduce S. mutans adhesion by competitively inhibiting the adsorption of S. mutans-binding salivary glycoproteins to hydroxyapatite (HA). The aim of this study was to develop antiadherence compounds for preventing dental biofilm development. We synthesized phosphorylated polyethylene glycol (PEG) derivatives and examined the possibility of surface pretreatment with them for preventing S. mutans adhesion in vitro and dental biofilm formation in vivo. Pretreatment of the HA surface with methacryloyloxydecyl phosphate (MDP)-PEG prior to saliva incubation hydrophilized the surface and thereby reduced salivary protein adsorption and saliva-promoted bacterial attachment to HA. However, when MDP-PEG was added to the saliva-pretreated HA (S-HA) surface, its inhibitory effect on bacterial binding was completely diminished. S. mutans adhesion onto S-HA was successfully reduced by treatment of the surface with pyrophosphate (PP), which desorbs salivary components from S-HA. Treatment of S-HA surfaces with MDP-PEG plus PP completely inhibited saliva-promoted S. mutans adhesion even when followed by additional saliva treatment. Finally, mouthwash with MDP-PEG plus PP prevented de novo biofilm development after thorough teeth cleaning in humans compared to either water or PP alone. We conclude that MDP-PEG plus PP has the potential for use as an antiadherence agent that prevents dental biofilm development.
By using a gnotobiotic rat model system to study the induction of protective immune responses by anti-idiotype (anti-id) vaccines specific for antibodies directed at the cariogenic microorganism Streptococcus mutans, it was shown that administration of such an anti-id vaccine provided partial protection against dental caries after challenge with virulent microorganisms. Protective effects were first demonstrated by direct parenteral administration of the anti-id vaccine into salivary gland regions, as determined by reductions in microbial colonization and caries scores. Subsequently, the anti-id was incorporated into liposomes and administered by gastric intubation. Immunization by this regimen also resulted in a significant reduction in caries as well as S. mutans colonization of the oral cavity, with concomitant increases in salivary immunoglobulin A anti-S. mutans antibodies. These data provide evidence that anti-id vaccines specifically targeted at the secretory immune system can induce protective immune responses to pathogens of mucosal surfaces.
The cariogenic bacterium Streptococcus mutans is an important dental pathogen that forms biofilms on tooth surfaces, which provide a protective niche for the bacterium where it secretes organic acids leading to the demineralization of tooth enamel. Lipids, especially glycolipids are likely to be key components of these biofilm matrices. The UA159 strain of S. mutans was among the earliest microorganisms to have its genome sequenced. While the lipids of other S. mutans strains have been identified and characterized, lipid analyses of UA159 have been limited to a few studies on its fatty acids. Here we report the structures of the four major glycolipids from stationary-phase S. mutans UA159 cells grown in standing cultures. These were shown to be monoglucosyldiacylglycerol (MGDAG), diglucosyldiacylglycerol (DGDAG), diglucosylmonoacylglycerol (DGMAG) and, glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG). The structures were determined by high performance thin-layer chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. The glycolipids were identified by accurate, high resolution, and tandem mass spectrometry. The identities of the sugar units in the glycolipids were determined by a novel and highly efficient NMR method. All sugars were shown to have α-glycosidic linkages and DGMAG was shown to be acylated in the sn-1 position by NMR. This is the first observation of unsubstituted DGMAG in any organism and the first mass spectrometry data for GPDGDAG.
Bacterium; glycolipids; lipidomics; dental pathogen
Streptococcus mutans is generally recognized as a causative agent of human dental caries. The production of mutacins (bacteriocins) by S. mutans is considered to be an important factor in the colonization and establishment of S. mutans in the dental biofilm. Two types of mutacins have been characterized: the lantibiotics and the non-lantibiotics. The lantibiotics generally have a wider spectrum of activity than the non-lantibiotics, which make them attractive targets for development into new antimicrobial modalities. The non-lantibiotics are much more prevalent among strains of S. mutans and play a significant role in both community and population level interactions in the dental biofilm. These interactions are directly mediated through the ComCDE two-component system and the newly characterized LytTR Regulation Systems HdrRM and BrsRM. These systems coordinate natural competence development and mutacin production as a means to acquire transforming DNA either by killing closely related streptococcal species in the vicinity of S. mutans, or through an altruistic suicide mechanism among a subpopulation of competent cells within the S. mutans community. As more S. mutans strains are sequenced, it is anticipated that additional mutacins with novel functions will be discovered, which may yield further insights into the ecological role of mutacins within the oral biofilm.
mutacin; Streptococcus mutans; bacteriocin
Antimicrobial peptides (AMPs) are naturally occurring, broad-spectrum antimicrobial agents that have recently been examined for their utility as therapeutic antibiotics. Unfortunately, they are expensive to produce and are often sensitive to protease digestion. To address this problem, we have examined the activity of a peptide mimetic whose design was based on the structure of magainin, exhibiting its amphiphilic structure. We demonstrate that this compound, meta-phenylene ethynylene (mPE), exhibits antimicrobial activity at nanomolar concentrations against a variety of bacterial and Candida species found in oral infections. Since Streptococcus mutans, an etiological agent of dental caries, colonizes the tooth surface and forms a biofilm, we quantified the activity of this compound against S. mutans growing under conditions that favor biofilm formation. Our results indicate that mPE can prevent the formation of a biofilm at nanomolar concentrations. Incubation with 5 nM mPE prevents further growth of the biofilm, and 100 nM mPE reduces viable bacteria in the biofilm by 3 logs. Structure-function analyses suggest that mPE inhibits the bioactivity of lipopolysaccharide and binds DNA at equimolar ratios, suggesting that it may act both as a membrane-active molecule, similar to magainin, and as an intracellular antibiotic, similar to other AMPs. We conclude that mPE and similar molecules display great potential for development as therapeutic antimicrobials.
The oral cavity is colonized by microorganisms growing in biofilms in which interspecies interactions take place. Streptococcus mutans grows in biofilms on enamel surfaces and is considered one of the main etiological agents of human dental caries. Candida albicans is also commonly found in the human oral cavity, where it interacts with S. mutans. C. albicans is a polymorphic fungus, and the yeast-to-hypha transition is involved in virulence and biofilm formation. The aim of this study was to investigate interkingdom communication between C. albicans and S. mutans based on the production of secreted molecules. S. mutans UA159 inhibited C. albicans germ tube (GT) formation in cocultures even when physically separated from C. albicans. Only S. mutans spent medium collected in the early exponential phase (4-h-old cultures) inhibited the GT formation of C. albicans. During this phase, S. mutans UA159 produces a quorum-sensing molecule, competence-stimulating peptide (CSP). The role of CSP in inhibiting GT formation was confirmed by using synthetic CSP and a comC deletion strain of S. mutans UA159, which lacks the ability to produce CSP. Other S. mutans strains and other Streptococcus spp. also inhibited GT formation but to different extents, possibly reflecting differences in CSP amino acid sequences among Streptococcus spp. or differences in CSP accumulation in the media. In conclusion, CSP, an S. mutans quorum-sensing molecule secreted during the early stages of growth, inhibits the C. albicans morphological switch.
The interplay between mucosal immune responses to natural exposure to mutans streptococci and the incorporation and accumulation of these cariogenic microorganisms in oral biofilms is unclear. An initial approach to explore this question would be to assess the native secretory immunity emerging as a consequence of Streptococcus mutans infection. To this end, we analyzed salivary immunoglobulin A (IgA) antibody to mutans streptococcal glucosyltransferase (Gtf) and glucan binding protein B (GbpB) and to domains associated with enzyme function and major histocompatibility complex (MHC) class II binding in two experiments. Salivas were collected from approximately 45-day-old Sprague-Dawley rats, which were then infected with S. mutans SJ32. Infection was verified and allowed to continue for 2 to 2.5 months. Salivas were again collected following the infection period. Pre- and postinfection salivas were then analyzed for IgA antibody activity using peptide- or protein-coated microsphere Luminex technology. S. mutans infection induced significant levels of salivary IgA antibody to Gtf (P < 0.002) and GbpB (P < 0.001) in both experiments, although the levels were usually far lower than the levels achieved when mucosal immunization is used. Significantly (P < 0.035 to P < 0.001) elevated levels of postinfection salivary IgA antibody to 6/10 Gtf peptides associated with either enzyme function or MHC binding were detected. The postinfection levels of antibody to two GbpB peptides in the N-terminal region of the six GbpB peptides assayed were also elevated (P < 0.031 and P < 0.001). Interestingly, the patterns of the rodent response to GbpB peptides were similar to the patterns seen in salivas from young children during their initial exposure to S. mutans. Thus, the presence of a detectable postinfection salivary IgA response to mutans streptococcal virulence-associated components, coupled with the correspondence between rat and human mucosal immune responsiveness to naturally presented Gtf and GbpB epitopes, suggests that the rat may be a useful model for defining mucosal responses that could be expected in humans. Under controlled infection conditions, such a model could prove to be helpful for unraveling relationships between the host response and oral biofilm development.
Streptococcus mutans is the major microbial pathogen associated with dental caries in children. The objectives of this study were to design and evaluate species-specific primers for the identification of S. mutans. Validation of the best primer set, Sm479F/R, was performed using 7 S. mutans reference strains, 48 ATCC non-S. mutans strains, 92 S. mutans clinical isolates, DNA samples of S. mutans-S. sobrinus or S. mutans-S. sanguinis, and mixed bacterial DNA of saliva samples from 33 18-month-old children. All of the S. mutans samples tested positive, and no PCR products were amplified from members of the other streptococci or non-streptococci strains examined. The lowest detection level for PCR was 10−2 nanograms of S. mutans DNA (approximately 4.6 × 103 copies) in the test samples. The results of our study suggest that the Sm479F/R primer pair is highly specific and sensitive for identification of S. mutans in either purified or mixed DNA samples.
Streptococcus mutans; PCR; species-specific primer; dental caries
The B subunit of cholera toxin (CTB) has been shown to augment mucosal responses to microbial virulence antigens, including those of Streptococcus mutans, which is the principal etiologic agent of dental caries. In the present study, the surface fibrillar protein antigen of S. mutans, antigen I/II (Ag I/II), was chemically coupled to CTB (Ag I/II-CTB), and the conjugate was examined for its effectiveness in inducing salivary immune responses protective against S. mutans infection. Weanling Fischer rats were given Ag I/II-CTB (50 micrograms) by the intranasal route and then orally infected with a virulent strain of S. mutans. Gnotobiotic or conventional rats were given two or three additional immunizations, respectively, at about 2-week intervals. One week after each immunization, individual serum, saliva, and fecal samples were collected and stored frozen until assayed for antibody activity to Ag I/II and cholera toxin (CT) by an enzyme-linked immunosorbent assay. The rats were sacrificed 1 week after the last immunization, when mandibles were also collected from individual rats for assessment of S. mutans levels in plaque and caries activity. Rats immunized only or both immunized and infected showed a salivary immunoglobulin A (IgA) anti-Ag I/II response which reached significantly (P < 0.05) higher levels than those seen in nonimmunized, infected controls. A salivary IgA anti-Ag I/II response was also seen in rats infected only with S. mutans. Essentially no salivary antibody activity to CT was detected. Some serum anti-Ag I/II and anti-CT responses were seen in immunized animals. Serum IgG anti-Ag I/II responses were seen in immunized, infected rats and also in infected-only rats, suggesting that the responses were a result of infection with S. mutans. The immunized and infected rats had significantly (P < 0.05) lower levels of S. mutans in plaque and lower caries activity than nonimmunized, infected rats. These results indicated that intranasal immunization of rats with Ag I/II-CTB induced a protective salivary immune response which was associated with a reduction in S. mutans colonization and S. mutans-induced dental caries.